Immobilized organophosphate-degrading enzymes and methods of making the same

ABSTRACT

The present application discloses immobilized enzymes and immobilized enzyme materials comprising a crosslinked organophosphate-degrading enzyme having a support material which includes a biomass material and/or a polymeric material. The resulting immobilized enzyme materials may be biodegradable. The present application also discloses methods of making and using the disclosed immobilized organophosphate hydrolase enzyme and enzyme materials.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority to U.S. ProvisionalPatent Application No. 61/558,758 filed on Nov. 11, 2011.

FIELD

The claimed technology relates generally to biocatalysts such as enzymesand more specifically to the immobilization of enzymes.

BACKGROUND

Immobilized biocatalysts have found applications in a variety ofindustries where specific chemical conversions are required. Specificlarge scale examples in the food and pharmaceutical industries includeimmobilized glucose isomerase for the conversion of glucose to fructoseand immobilized penicillin acylase for the preparation of derivatives ofpenicillin. Immobilized enzymes can be used for large scalebioremediation such as the destruction of undesirable chemicalcompounds. Another application involves small scale immobilized enzymesused in diagnostic reactions wherein the biocatalyst facilitates achemical reaction which produces a detectable chemical moiety or otherchemical change. More applications for immobilized enzymes will likelybe developed in the future.

Immobilized biocatalysts (enzymes) provide advantages over biocatalystsin solution such as those provided in a liquid form or in a dry form tobe dissolved in a liquid. Immobilized enzymes can be easily separatedfrom the solution and thus permit the reuse of these enzymes hencereducing the cost of the enzyme in the production of a product. In thetwo cases cited above, immobilized enzymes are used in a packed columnwith the reactant solution pumped through the column and the desiredchemical conversion accomplished when the solution leaves the column. Inthis process, the enzymes are used multiple times at high concentrationsto achieve efficient usage of the enzymes.

In addition to ease of separation, immobilized enzymes can provide otherpotential advantages including greater thermal stability, pH stability,and the like during storage as well as during usage. Immobilizedbiocatalysts also can provide greater activity in various solventscompared to the enzymes in solution. There are often advantages to thephysical form because immobilized biocatalysts can be provided in agranular form which reduces dust and improves handling of the product.As with a dried enzyme, the activity of the immobilized enzyme per massof material can be varied as desired for a particular application.

An immobilized enzyme may be composed of the enzyme material combinedwith a solid matrix to which the enzyme is, attached or held by chemicalor physical means. The enzyme may be attached to the surface of thematrix, or if the matrix is porous enough to permit diffusion of thesubstrate, the enzyme may be distributed throughout the matrix. Thereare many methods proposed for the immobilization of biocatalystsincluding the entrapment of the enzyme in gel, the covalent,hydrophobic, electrostatic, and other methods for attachment of theenzyme to an inorganic or organic solid. The cross linking of the enzymewith whole cells, enzyme crystals, etc may be accomplished usingreagents such as glutaraldehyde (GA) and polyethylenimine (PEI).

The conversion of a biocatalyst to an immobilized form may require moreprocessing and hence may increase the cost of production for that enzymeform. Any increased cost must be compensated by increased productivity,shelf life, use in solvents, or some other advantage. Therefore, thecost of immobilization is an important consideration in the productionof an immobilized enzyme. A lower cost for immobilization hence providesfor a more economical usage of the immobilized enzyme.

Of the methods proposed for the immobilization of the enzymes, the useof cross linking whole cells is one of the most cost-effective. Thismethod utilizes the whole cells are those of the microorganism used tomake the enzyme and hence are available for no cost or even a negativecost if there is a cost associated with the disposal of the cell mass.

Various immobilization processes have been suggested for enzymesincluding attachment to silica supports. This process involves thefermentation of microorganism for the production of the enzyme, theseparation (or purification) of the enzyme from the biomass, thechemical attachment or encapsulation of the enzyme to the matrix, andthe production of the final product form. These immobilizations have thedisadvantage of incurring the cost of the matrix to which the enzyme isattached. Further the separation of the enzyme from the fermentationbiomass involves some loss of enzyme and enzyme activity, as well asassociated purification processing costs. These disadvantages areovercome by the immobilization of the enzyme directly with the biomassused to produce the enzyme.

Some enzyme substrates have limited solubility in water and have bettersolubility in organic solvents or mixtures of organic solvents andwater. For example, due to these properties, organic solvent solutionsare often used to wash away organophosphorus compounds or organicsolvents are used in the processing of triglycerides. Soluble enzymesare often deactivated or rendered inert in organic solvents or mixtures;however, immobilized enzymes can maintain some activity in solventscompared to soluble enzymes. Hence immobilized enzymes have the capacityto react with the higher concentrations of their substrates in suchsolvents.

Application of immobilized enzymes may require the disposal of theenzyme after usage. For example, the immobilized enzyme used in a columnreactor would require disposal after use. Also, some immobilized enzymescan be considered for use directly in the environment. For example,immobilized OPH enzyme could be dispersed on an agricultural field fordecontaminating a pesticide or other organophosphorus compound.

SUMMARY

The claimed technology is set forth in the claims below, and thefollowing is not in any way to limit, define or otherwise establish thescope of legal protection. In general terms, the disclosed technologyrelates to immobilized enzymes and methods of producing the same.

One aspect of the disclosed technology provides a range of immobilizedenzymes that utilize one or more biomass materials as components of asupport matrix. The disclosed technology therefore minimizes cost andsimplifies production. Advantageously, such immobilized enzymes arestable and able to operate over a range of conditions, in particular inthe presence of a range of solvents, over a range of pH values, and atthe temperatures encountered in the field. The immobilized enzymes andimmobilized enzyme materials are, in some instances, also biodegradable.Specific immobilized enzymes are provided which are able to transformorganophosphates and related toxic materials into less harmfullharmlessmaterials and optionally these are capable of biodegradation to furthereffect and simplify remediation efforts. Methods for the preparation andutilization of these immobilized enzymes are also provided.

In general, the disclosed technology provides an immobilized enzymematerial. The material may be an immobilized organophosphate-degradingenzyme material. The enzyme material may comprise a crosslinked enzymehaving a support matrix that includes a biomass material. The biomassmaterial may be different from the biomass material the enzyme wasderived from. The crosslinked enzyme may be formed by reacting theenzyme with at least two polyfunctional materials.

Enzymes that can be incorporated into the immobilized enzymes of thedisclosed technology include, but are not limited to, enzymes from aclass selected from the group consisting of Oxidoreductases,Transferases, Hydrolases, Lyases, Isomerases, and Ligases.Organophosphate-degrading enzymes such as organophosphate hydrolase(OPH), organophosphorus acid anhydrolase (OPAA),diisopropyl-fluorophosphatase (DFPase), and the like may be used.Enzymes such as lactase, glucose isomerase, peroxidase, alcoholdehydrogenase, xylanase, pyruvate decarboxylase, catalase, invertase,phytase, amyloglucosidase, glucose oxidase, penicillin acylase, and thelike may also be mentioned. The immobilized enzymes of the disclosedtechnology may exhibit improved temperature stability compared to thefree enzyme. Optionally, the disclosed technology may be adapted andapplied to combinations of two or more enzymes.

The disclosed technology therefore provides the possibility of low costenzyme products, where the enzyme may be organophosphate hydrolaseand/or other enzymes. These products can be used for the destruction oforganophosphorus compounds and for other chemical transformations. Thedisclosed technology provides an enzyme product that is stable in bothstorage and usage conditions. Further, the disclosed technology providesan enzyme product where the enzyme is active in solvents in which someorganophosphorus compounds are more soluble or in which other chemicaltransformations can be brought about. In addition, the disclosedtechnology provides a range of immobilized enzyme products fororganophosphorus decomposition as well as other enzyme applications thatrequire the enzyme to be disposed of or left in the environment todecompose. These products are suitably biodegradable.

In the disclosed technology, the immobilized enzyme materials canfunction over a substantially broader pH range than the free enzyme, inthe presence of a range of solvents, and at higher temperatures than thefree enzyme.

The biomass material that is utilized in the support material mayinclude biomass that is different from the biomass material the enzymewas derived from. In this regard it may include a combination of biomassthat is different from the biomass material the enzyme was derived fromand biomass that is the same as the biomass material the enzyme wasderived from thereof, provided at least some of the biomass is differentfrom the biomass the enzyme was derived from.

There are benefits in using biomass that is different from the biomassthe enzyme was derived from. In this regard, one advantage is that thebiomass that is different from the biomass the enzyme was derived frommay be a waste product that otherwise would need to have been disposedof. Another advantage is that the characteristics of that biomass can beselected depending on the intended use of the immobilized enzyme. Forexample, the immobilized enzyme can be made biodegradable. This isuseful when the immobilized enzyme is used to clean up environmentalmishaps, including some forms of pesticides and chemical warfare agents,and in manufacturing applications where later disposal of theimmobilized enzyme becomes necessary. For example, treatment of a fieldcontaminated with an organophosphate pesticide can be decontaminated byapplying a biodegradable form of the immobilized enzyme over the field'ssurface. Once the degradation is complete, the biodegradable immobilizedenzyme simply degrades over time, making further clean-up/removalunnecessary.

In some examples, the physical properties of the final immobilizedenzyme material can be modified through the choice of biomass. Forexample, the material can be made more or less rigid to enhance itssuitability for a variety of reactor configurations. A more rigidparticle would be advantageous in a packed bed reactor configuration.Similarly, immobilized enzyme material properties such as porosity,hydrophobicity, and solubility may be modified through the selection ofbiomass material to suit specific reaction conditions. In general,incorporating additional biomass, different from that which the enzymeswas derived, provides additional options for specifically adapting theimmobilized enzyme material compared to whole cell immobilizations thatare limited to the properties of the biomass from which the enzyme wasderived.

Suitable support materials may have a plurality of amine groups or otherfunctional groups capable of reacting with the crosslinking material.Support materials can include one or more biomass materials, such aschitin, Aspergillus niger cells, wool and the like. One type of suitablebiomass materials has a plurality of functional groups capable ofreacting with the crosslinking material. Biomass materials containingamino groups thereon, such as for example a polyamine, have been used.Examples of such suitable biomass support materials include, but are notlimited to, Aspergillus niger cells, yeast cells, cellulose, dextran,starch agar, alginate, carrageenans, collagen, gelatin, albumin, andferritin. Other biomass materials such as cotton and wool can optionallybe included, provided at least some support material having a pluralityof functional groups is provided to support crosslinking. Supportmaterials may also include synthetic and/or polymeric materials such aspolyethylenimine, chitosan, polypyrrole, or other suitable material.Optionally, the support material may comprise a combination of one ormore biomass materials and/or one or more polymeric support materials.Suitable polymeric support materials can include polyamines, such as forexample polyethylenimine, polypyrrole, a protein, gelatin, and the like.In general, suitable biomass materials can include biomass materialsgenerally free of functional groups and minimally involved in thecrosslinked structure, biomass materials having a plurality offunctional groups that are involved with the necessary crosslinking, andcombinations thereof.

Suitable crosslinking materials include polyfunctional groups capable ofreacting with the support material. Suitable crosslinking materials mayalso include polyfunctional groups capable of reacting with the supportmaterial, such as, for example, glutaraldehyde, di-aldehyde, an organicdi-acid, disuccinimidyl suberate, dimethyl pimelimidate, dimethyladipimidate, cyanuric chloride, succinic acid, hexamethylenediisocyanate, and the like. Other reagents capable of crosslinking toamines may also be contemplated.

Immobilized enzymes that utilize a biomass material for the supportmatrix are optionally biodegradable. In other examples, the biomassmaterial is insoluble. The immobilized enzymes of the disclosedtechnology may therefore be biodegradable. Suitable biodegradablesupport materials include, but are not limited to, processed orunprocessed exoskeleton (mineral or organic), bone, chitin (soluble orinsoluble), Aspergillus niger cells and the like.

In applications where the immobilized enzyme is applied over a largearea open to the environment it can be advantageous for the immobilizedenzyme to be biodegradable. Thus, the embodiments of the disclosedtechnology where the immobilized enzyme is biodegradable has utility fora variety of enzymes requiring disposal or enzymes that are dispersed inthe environment.

The immobilized enzyme material can additionally include a solvent. Thismay be a solvent adapted to dissolve the material sought to betransformed. Solvents can include, but are not limited to, hexane,toluene, methanol, dimethyl sulfoxide, and the like. The immobilizedenzymes may exhibit improved temperature stability compared to the freeenzyme.

In general, the disclosed technology also provides a method forpreparing such an immobilized enzyme material. In one example, themethod includes the steps of providing an enzyme, a support material anda crosslinking material, and combining the support material and thecrosslinking material with an aqueous suspension of the enzyme. Thematerials may be combined in any order; in some instances, the supportmaterial is added before the crosslinking material, whereas in otherinstances, the crosslinking material is added before the supportmaterial, in order to maximize activity. The step of combining thematerials may suitably result in a flocculated product. The product canbe used directly or isolated, dried, and formulated into particleshaving a desirable size and shape, depending on the intendedapplication. The support material may include a biomass supportmaterial. Support materials other than biomass support materials canadditionally be provided and added to the aqueous suspension of theenzyme. Optionally, the crosslinking material may be provided in asuitable solvent, such as water, alcohol, DMSO, and the like.

The immobilized enzyme material thus fowled may be subjected to elevatedtemperatures. Subjecting the immobilized enzyme to elevated temperatureshas generally increased the activity of the immobilized enzymes comparedto a similar immobilized enzyme not subject to a treatment at elevatedtemperatures.

In general, the disclosed technology also provides a method forutilizing the immobilized enzyme material to transform a materialsusceptible to enzymatic transformation. The immobilized enzyme materialmay be provided in biodegradable form. In one example, the methodinvolves providing the immobilized enzyme material, wherein the enzymeis in a form suitable for contacting the material susceptible toenzymatic transformation and adapted to effect the desiredtransformation, and contacting the immobilized enzyme material with thematerial susceptible to enzymatic transformation. In some examples suchcontacting involves dissolving the material susceptible to enzymatictransformation in a solvent to form a solution, and contacting theimmobilized enzyme material with the solution. In applications involvingenvironmental remediation, contacting can involve dispersing theimmobilized enzyme material over the soil and depending on moisture inthe soil to act as a solvent to dissolve the material susceptible toenzymatic transformation. In other examples where the materialsusceptible to enzymatic transformation is not soluble in water or wateris not available, a solvent may need to be supplied. In other examples,the desired transformations may be carried out in a column, a vessel, orother reactor in which the immobilized enzyme material and the materialsusceptible to enzymatic transformation are combined with a solvent inwhich the material susceptible to enzymatic transformation has at leastsome solubility. In some embodiments the transformation involvestransforming or degrading a material, which may be selected frompesticides, chemical warfare agents, nerve agents, and the like, intoless harmfullharmless moieties. In general, the transformation may be adecomposition to a less harmful/harmless moiety or may be a conversionto a desired chemical compound.

A first aspect of the present disclosure is to provide immobilizedorganophosphate-degrading enzymes. In one example, a crosslinkedorganophosphate-degrading enzyme (ODE) was demonstrated byorganophosphate hydrolase (OPH) formed by reacting the OPH with asupport material and a crosslinking material, although in other examplesother ODE may be used. Suitable support materials optionally have aplurality of amine groups. Support materials can include biomassmaterials such as chitin, Aspergillus niger cells, wool and the like. Inaddition, support materials can be polyamines, such as polyethylenimine,polypyrrole, a protein, gelatin, and the like. Suitable crosslinkingmaterials may include polyfunctional groups capable of reacting with thesupport material such as for example glutaraldehyde, disuccinimidylsuberate, dimethyl pimelimidate, and the like.

In a further example, the crosslinked OPH material can involve a supportmatrix that includes a biomass material. The biomass material can be thebiomass material that was used to generate the OPH, another biomassmaterial, or a combination thereof. Examples of suitable supportmaterials that are biomass materials include, but are not limited tochitin, Aspergillus niger cells, wool and the like. Immobilized enzymesthat utilize a biomass material for the support matrix and the supportmaterial are optionally biodegradable. In other examples, the biomassmaterial is insoluble.

In still a further example, the crosslinked OPH material canadditionally include a solvent, optionally a solvent having a solventadapted to dissolve the material sought to be transformed. Solvents caninclude, but are not limited to hexane, toluene, methanol, dimethylsulfoxide, and the like. The immobilized OPH enzymes may exhibitimproved temperature stability compared to the free enzyme.

A further aspect of the present disclosure includes immobilized enzymeshaving a support matrix that includes a biomass material that isdifferent from the one the enzyme was derived from. In one such example,the immobilized enzyme can be formed by reacting an enzyme with asupport material and a crosslinking material where the support materialincludes a biomass material. Biomass material utilized can includebiomass that is different from the biomass material the enzyme wasderived from, the same, or a combination thereof, provided at least someof the biomass is different from the biomass the enzyme was derivedfrom. One type of suitable biomass materials has a plurality offunctional groups capable of reacting with the crosslinking material.Biomass materials containing amino groups thereon, such as for example apolyamine, have been used. Examples of such suitable biomass supportmaterials include, but are not limited to Aspergillus niger cells, yeastcells, cellulose, dextran, starch agar, alginate, carrageenans,collagen, gelatin, albumin, and ferritin. Other biomass materials suchas cotton and wool can optionally be included, provided at least somesupport material having a plurality of functional groups is provided tosupport crosslinking. Suitable support materials can also includepolyamines, such as for example polyethylenimine, polypyrrole, aprotein, gelatin, and the like. Suitable crosslinking materials mayinclude polyfunctional groups capable of reacting with the supportmaterial. Crosslinking materials suitable for reacting with supportmaterials containing amines may include glutaraldehyde, disuccinimidylsuberate, dimethyl pimelimidate, and the like. Some examples of enzymesthat can be incorporated into these immobilized enzymes include enzymesfrom a class selected from the group consisting of Oxidoreductases,Transferases, Hydrolases, Lyases, Isomerases, and Ligases. Theimmobilized enzymes described above may exhibit improved temperaturestability compared to the free enzyme.

A still further aspect of the present disclosure includes immobilizedenzymes which are biodegradable. Such biodegradable enzymes include acrosslinked enzyme having a support matrix derived from a supportmaterial that includes a biomass and a crosslinking material. Suitablebiomass support materials optionally have a plurality of functionalgroups capable of reacting with the crosslinking material. Some biomassmaterials containing amino groups thereon, such as for example apolyamine, have also been used. In other examples, the biomass materialused is soluble or insoluble in water as desired. Still other examplesof such suitable biomass support materials include, but are not limitedto Aspergillus niger cells or other bacterial cellular materials, yeastcells or other fungal cellular materials, cellulose, dextran, starchagar, alginate, carrageenans, collagen, gelatin, albumin, and ferritin.Other biomass materials such as cotton and wool can optionally beincluded. Suitable crosslinking materials may optionally includepolyfunctional groups capable of reacting with the support material. Inother examples, crosslinking materials which are heterofunctional, thatis they contain two or more different functional groups, may be used.Some crosslinking materials suitable for reacting with support materialscontaining amines include glutaraldehyde, disuccinimidyl suberate,dimethyl pimelimidate, and the like. Some enzymes that can beincorporated into these immobilized enzymes include enzymes from a classselected from the group consisting of Oxidoreductases, Transferases,Hydrolases, Lyases, Isomerases, and Ligases. The biodegradableimmobilized enzymes may exhibit improved temperature stability comparedto the free enzyme.

A still further aspect of the present disclosure involves a method forpreparing an immobilized enzyme in which at least a portion of thesupport material is a biomass material. In one example; the methodincludes the steps of providing an enzyme, a biomass support materialand a crosslinking material, adding the biomass support material and thecrosslinking material to an aqueous suspension of the enzyme withagitation to form a flocculent. An aqueous suspension can include anysuspension which includes at least some water. Biomass materials otherthan biomass support materials can additionally be provided and added tothe aqueous suspension of the enzyme. Support materials can additionallyinclude a polyamine selected from the group consisting ofpolyethylenimine, polypyrrole, polyethylenediamine; a polyethylenimine(such as, for example, polydiethylenetriamine, polytriethylenetetramine,polypentaethylenehexamine or polyhexamethylenediamine);polymethylenedicyclohexylamine; polymethylenedianiline;polytetraethylenepentamine; polyphenylenediamine, blends of two or moreof these polyamine compounds, and the like. A further example includes amethod for preparing a biodegradable immobilized enzyme which includesthe step of providing one or more support materials and a crosslinkingagent that are biodegradable. Suitable biodegradable support materialsinclude, but are not limited to processed (by grinding, pulverizing, orother mechanical processes) or unprocessed exoskeleton (mineral ororganic), bone, chitin (soluble or insoluble), Aspergillus niger cellsand the like. Suitable crosslinking materials include, but are notlimited to glutaraldehyde, a di-aldehyde, an organic di-acid,disuccinimidyl suberate, and dimethyl pimelimidate, polyfunctionalaldehydes, polyfunctional organic halides, polyfunctional anhydrides,polyfunctional azo compounds, polyfunctional isothiocyanates,polyfunctional isocyanates, blends of two or more of these aminereactive materials, succindialdehyde, terephthaldehyde,bis-diazobenzidine-2,2′-disulfonic acid,4,4′-difluoro-3,3′-dinitrodiphenylsulfone,diphenyl-4,4′-dithiocyanate-2,2′-disulfonic acid,3-methoxy-diphenylmethane-4,4′-diisocyanate,toluene-2-isocyanate-4-isothiocyanate, toluene-2,-4-diisothiocyanate,diazobenzidine, diazobenzidine-3,3′-dianisidine, N,N′-hexamethylenebisiodoacetamide, hexamethylene diisocyanate, cyanuric chloride,1,5-difluoro-2,4-dinitrobenzene, blends or two or more of these aminereactive materials, and the like. Optionally, the crosslinking materialmay be provided in a suitable solvent such as water, alcohol, DMSO, andthe like. The method can be utilized to form immobilized enzymes from aclass selected from the group consisting of Oxidoreductases,Transferases, Hydrolases, Lyases, Isomerases, and Ligases. Subjectingthe immobilized enzymes described above to elevated temperatures afterisolation can increase the immobilized enzymes activity compared to asimilar immobilized enzyme not subject to a treatment at elevatedtemperatures.

A still further aspect of the present disclosure involves a method forutilizing a biodegradable enzyme to transform a material susceptible toenzymatic transformation. In one such example, the method involvesproviding a biodegradable enzyme in a form suitable for contacting thematerial and adapted to effect the desired transformation, andcontacting the biodegradable immobilized enzyme with the materialsusceptible to enzymatic transformation. Optionally such contactinginvolves dissolving the material susceptible to enzymatic transformationin a solvent to form a solution, and contacting the biodegradableimmobilized enzyme with the solution. In applications involvingenvironmental remediation, contacting can involve dispersing thebiodegradable immobilized enzyme over the soil and depending on moisturein the soil to act as a solvent to dissolve the material susceptible toenzymatic transformation. In other examples where the materialsusceptible to enzymatic transformation is not soluble in water or wateris not available, a solvent may be supplied.

In particular organophosphate compounds are potent neurotoxins commonlyused as pesticides, insecticides, and as chemical warfare agents.Although organophosphate use as a chemical warfare agent has beenrestricted by international treaties; use of these compounds asagricultural and domestic pest controls present legitimate concernsabout contamination of soil and water systems, along with remediation ofcontaminated facilities and containers. Many organophosphates can bedecomposed with chemical decontaminants such as sodium hydroxide,potassium hydroxide, hypochlorite, and hydrogen peroxide or an area canbe treated with detergent and water to remove the contamination.Although each of these methods is effective at treating contamination toan extent, there are also secondary issues that must be considered inthe application. For example, several of these materials are corrosivein nature and all waste products from the treatments must still behandled as hazardous waste.

In order to retain the effectiveness of a chemical treatment and improveupon the waste handling aspects of a treatment process the utilizationof Organophosphate Degrading Enzymes (ODE) for decontamination oforganophosphates is considered. ODE is a general term to describeenzymes which are capable of catalyzing the hydrolysis of a wide rangeof organophosphate triesters and organophosphofluoridates.Aryldialkylphosphatases (E.C. 3.1.8.1) and diisopropyl-fluorophosphatase(E.C. 3.1.8.2) have been shown to be capable of cleaving P—O, P—F, P—S,P—CN bonds making them reactive against a wide variety oforganophosphate pesticides such as: acephate, coumaphos, demeton,diazinon, dursban, malathion, paraoxon, parathion, methyl paraoxon, andmethyl parathion; and chemical warfare agents including: sarin, soman,and VX.

In other examples, the desired transformations may be carried out in acolumn, a reaction bed, a vessel, or other reactor in which thebiodegradable immobilized enzyme and the material susceptible toenzymatic transformation are combined with a solvent in which thematerial susceptible to enzymatic transformation has at least somesolubility. In both of the above examples, the biodegradable immobilizedenzymes can function over a substantially broader pH than the freeenzyme, in the presence of a range of solvents, and at highertemperatures than the free enzyme. Examples of suitable solventsinclude, but are not limited to hexane, toluene, methanol, dimethylsulfoxide, and the like.

Further objects, embodiments, forms, benefits, aspects, features andadvantages of the disclosed technology may be obtained from thedescription, drawings, and claims provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing absorbance vs. time data of OPH enzyme in cellslurry.

FIG. 2 is a graph showing absorbance vs. time data of OPH enzyme in animmobilized form.

FIG. 3 is a graph showing absorbance vs. time data for OPH enzyme incell slurry and in immobilized forms with different particle sizes afterexposure to 52.5° C. temperature for four hours.

FIG. 4 is a graph showing absorbance vs. time for OPH in a cell slurryin methanol solutions.

FIG. 5 is a graph showing absorbance vs. time for forms of immobilizedOPH in methanol solutions.

FIG. 6 is a graph showing absorbance vs. time for forms of immobilizedOPH with different particle sizes in methanol solutions.

FIG. 7A is a graph showing absorbance vs. time data for OPH in a cellslurry after various amounts of time exposed to 65° C.

FIG. 7B is a graph showing absorbance vs. time data for an immobilizedform of OPH after various amounts of time exposed to 65° C.

FIG. 8 is a graph showing absorbance vs. time data for soluble OPHenzyme.

FIG. 9 is a graph showing absorbance vs. time data for immobilized formsof OPH enzymes.

FIG. 10 is a graph showing absorbance vs. time data for OPH in a cellslurry in 10% methanol solution.

FIG. 11 is a graph showing absorbance vs. time data for OPH in a cellslurry in 20% methanol solution.

FIG. 12 is a graph showing absorbance vs. time data for OPH in a cellslurry in 30% methanol solution.

FIG. 13 is a graph showing absorbance vs. time data for OPH in a cellslurry in 40% methanol solution.

FIG. 14 is a graph showing absorbance vs. time for immobilized OPH in10% methanol solution.

FIG. 15 is a graph showing absorbance vs. time for immobilized OPH in20% methanol solution.

FIG. 16 is a graph showing absorbance vs. time for immobilized OPH in30% methanol solution.

FIG. 17 is a graph showing absorbance vs. time for immobilized OPH in40% methanol solution.

FIG. 18 is a graph showing the change in activity of free OPH enzymeover time with exposure to ethylene glycol solutions relative to itsactivity in a solvent-free solution.

FIG. 19 is a graph showing the change in activity of the OPH-7immobilized form of the OPH enzyme over time with exposure to ethyleneglycol solutions relative to its activity in a solvent-free solution.

FIG. 20 is a graph showing the change in activity of the OPH-14immobilized form of the OPH enzyme over time with exposure to ethyleneglycol solutions relative to its activity in a solvent-free solution.

FIG. 21 is a graph showing the change in activity of the OPH-15immobilized form of the OPH enzyme over time with exposure to ethyleneglycol solutions relative to its activity in a solvent-free solution.

FIG. 22A is a graph showing absorbance vs. time data for Free PO aftervarious amounts of time exposed to 65° C. as either dry crystals or assuspended in buffer solution.

FIG. 22B is a graph showing absorbance vs. time data for an immobilizedform of PO after various amounts of time exposed to 65° C. either as adry powder or suspended in a buffer solution.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thedisclosed technology and presenting its currently understood best modeof operation, reference will now be made to the embodiments illustratedin the drawings and specific language will be used to describe the same.It will nevertheless be understood that no limitation of the scope ofthe disclosed technology is thereby intended, with such alterations andfurther modifications in the illustrated device and such furtherapplications of the principles of the disclosed technology asillustrated therein being contemplated as would normally occur to oneskilled in the art to which the disclosed technology relates.

The disclosed technology addresses the problem of providing: (a)immobilized enzymes capable of promoting a range of desirabletransformations that have a support matrix that includes a biomassmaterial; (b) methods for the preparing these immobilized enzymes, and(c) methods for utilizing the immobilized enzymes. The immobilizedenzymes of this disclosure have reduced material costs, good thermalstability, good activity in the presence of a range of solvents, andover a wide range of pH's, and can be produced to exhibitbiodegradability. Suitable biomass materials can include biomassmaterials generally free of functional groups and minimally involved inthe crosslinked structure, biomass materials having a plurality offunctional groups that are involved with the necessary crosslinking, andcombinations thereof. One example involves a crosslinked immobilizedenzyme that includes organophosphate hydrolase, and is capable oftransforming or degrading a range of pesticides, nerve agents, and thelike into less harmful/harmless moieties. Forms of the immobilizedorganophosphate hydrolase can be biodegradable.

The general methods for preparing the immobilized enzymes of thispresent disclosure involve providing the enzyme, the biomass material(when utilized), the support material, and the crosslinking material,suspending the enzyme in an aqueous medium, and combining the othermaterials provided to form a flocculent that can be isolated by standardmethods. In some instances, the support material should be added beforethe crosslinking material, whereas in other instances, the crosslinkingmaterial should be added before the support material in order tomaximize activity. The aqueous flocculent can be used directly orisolated, dried, and formulated into particles having a desirable sizeand shape, depending on the intended application. The activity ofcertain immobilized enzymes was significantly and surprisingly increasedupon heating the solid at an elevated temperature.

The following defined terms are utilized in the present disclosure andcarrying with them the meanings specifically described herein.

Support Matrix: One or more materials used to form a particle to impartmechanical and enzymatic stability upon an enzyme that can includewithin it, crosslinkers, reactive polymers, support materials, andbiomass.

Biomass: Material produced by a biological source such asmicroorganisms, plants, and/or other living organisms such as mollusks,insects, or crustaceans, or portions of organisms such exoskeletonsand/or shells (both organic and mineral-based). Such materials may bemechanically processed (grinding, pulverizing, and the like), but notchemically treated or processed.

Support Material: Materials used individually or in combination in aSupport Matrix, such as Biomass, synthetically manufactured polymers,and/or other non-organic materials.

Crosslinking Agent: chemical reagents containing two or more reactiveends that are used to covalently bond specific functional groups onenzymes and support materials, for instance, attaching amine groups withaldehydes.

Reactive Polymers: includes polymers with reactive pendant groups whichcan react with the crosslinkers.

Immobilization Procedures

The following examples are intended to be illustrative of the presentdisclosure and are not intended to otherwise limit the disclosure in anymanner As demonstrated by the examples that follow, enzymes from a widerange of enzyme classes that include Oxidoreductases, Transferases,Hydrolases, Lyases, Isomerases, and Ligases can be utilized according tothis present disclosure. The ODE enzymes used in the following exampleswere immobilized using host cells as either a cell slurry or lysed cellmaterial. In some examples, purified enzymes were used. Supportmaterials may also include synthetic and/or polymeric materials such aspolyethylenimine, chitosan, polypyrrole, or other suitable material.Optionally, the support material may comprise a combination of one ormore biosupport materials and/or polymeric support materials. Crosslinking comprised polyfunctional materials such as glutaraldehyde (GA),disuccinimidyl suberate (DSS), dimethyl pimelimidate (DMP), dimethyladipimidate (DMA), cyanuric chloride (CyC), succinic acid (SA),hexamethylene diisocyanate (HDI), and/or other reagents capable ofcrosslinking to amines.

An enzyme is provided (isolated or contained in myceliuma mixture whichmay contain other proteins or a cell slurry) and suspended in an aqueousbuffer solution. A support is added with stirring which is continued forabout 1 hour until the solution is well mixed. Optionally, a volume ofadditional deionized water may be added as desired. A crosslinker isadded with continued agitation to cause the immobilized enzyme toflocculate. The solid can be isolated and dried utilizing standardprocedures. This is but one example of a general immobilizationprocedure. In other examples, the components may be added in a differentorder, two or more may be added simultaneously, and/or the steps may beperformed over a longer or shorter period of time. The followingexamples are provided for illustrative purposes, and are not intended tolimit the disclosed technology in any manner.

Immobilization Process for Organophosphate Hydrolase

In the following examples, the OPH enzyme used is of the varietydisclosed in U.S. patent application Ser. No. 12/241,574 ('574application) titled Differentially Fluorescent Yeast Biosensors for theDetection and Biodegradation of Chemical Agents, which was filed on Sep.30, 2008. The methods and techniques described herein may also beapplied to other varieties of OPH and/or to varieties of OPH producedusing methods other than those disclosed in the '574 application. Themethods and techniques described herein may also be applied to enzymesother than OPH. Organophosphate-degrading enzymes such as otheraryldialkylphosphatases (E.C. 3.1.8.1) anddiisopropyl-fluorophosphatases (E.C. 3.1.8.2) and the like may also beused. In some examples, an organophosphate-degrading enzyme is one whichacts on one or more bonds connecting a phosphorous atom with a molecule,although the disclosed techniques may also be applied to enzymes whichdegrade or break down organophosphates using different mechanisms.Optionally, the techniques and methods disclosed herein may also beadapted and applied to combinations of two or more enzymes. In otherexamples the methods and techniques disclosed herein may be adapted andapplied to two or more enzymes individually and then the resultingimmobilized enzymes may be combined as desired for a particularapplication.

In one example, the enzyme is immobilized using biomass which waspreviously used to produce the enzyme. In this particular example, theenzyme is produced by a fermentation which generates a fermentationbroth containing the enzyme and the biomass of the organisms whichcreated the enzyme. The broth is removed from the fermenter and thesolid material is dried and optionally processed to yield particles of adesired size. Optionally, small molecules may be removed from thesolution prior to drying. Additionally, enzyme cofactors, substancesthat form a solid material containing the biomass and enzyme, oradditional substances may be added to the fermenter prior to removal ofthe fermentation broth, and/or directly to the solid material eitherbefore or after drying as desired.

The immobilized enzyme formed by such a process is may be less expensivethan other immobilization processes because there is no cost for a solidmatrix material used to support the enzyme. Also, less enzyme and enzymeactivity is lost when the enzyme is retained with the biomass.

This immobilized enzyme may demonstrate greater thermal stability thanthe free enzyme. Hence it can withstand greater temperature fluctuationsin both storage and usage and still maintain activity. The immobilizedenzyme may also demonstrate greater activity in solvent solutions.Increased activity in solvents other than water is beneficial becausesome organophosphorus compounds are more soluble in solvents other thanwater. Solvents that might be considered are alcohol/water mixtures suchas 20 percent methanol in water as well as other solvents.

Another aspect of the present disclosure is to provide an inexpensiveimmobilized enzyme which exhibits increased biodegradation under avariety of environmental conditions. In some examples, most or all ofthe chemical components which comprise an immobilized enzyme willexhibit increased biodegradation in a biodegradable immobilized enzymeproduct. In one example, an enzyme is immobilized using the biomasswhich was used to produce the enzyme where the biomass itself isbiodegradable. In other examples, biomass from other fermentations couldbe used to immobilize a soluble enzyme first produced using othermethods. In still other examples, the biomass material used is insolubleor has limited solubility in water.

The following examples describe methods for immobilizing enzymes usingthe enzyme OPH. The use of one specific enzyme (OPH) in the examples wasdone for the sake of convenience. The disclosed methods and techniquesfor immobilizing enzymes are not limited to OPH. It is understood thatone of ordinary skill in the art would be able to adapt the disclosedmethods and techniques to immobilize a wide variety of other enzymesincluding, but not limited to, lactase, glucose isomerase, catalase,invertase, phytase, amyloglucosidase, glucose oxidase, penicillinacylase, and the like.

Immobilization of Enzyme-Containing Cells Cell Growth, EnzymeProduction, Initial Preparation

In one example, production of OPH from engineered E. coli begins withthe preparation of a culture stock solution. The initial solution, 125mL, consisting of 25 g/L LB Broth, is sterilized at 121° C. for 15minutes. After sterilization it is allowed to cool and 50 μg/mL ofkanamycin and 50 μg/mL of chloramphenicol are aseptically added to thestock solution. A seed culture is prepared from 2 mL of this stocksolution and 1 colony of E. coli harvested from an agar plate that waspreviously streaked or inoculated with the desired E. coli organism.This 2 mL solution is incubated for 24 hours at 37° C. on a shake tableat 150 RPM. After the incubation period has ended, seed culture solutionis added in a sterile manner at 1% v/v to a larger volume (20 mL) of theculture stock solution, identified as the starter culture. This starterculture is then incubated for 24 hours at 37° C. on a shake table at 150RPM.

The fermentation broth solution, consisting of 15 g/L dextrose, 10 g/Lyeast extract, and 5 μg/L Pharmamedia in tap water is introduced intothe 14 liter fermentation vessel (10 liter working volume) New BrunswickBioFlo 3000 fermenter. This solution is sterilized in the fermentationvessel at 121° C. for 15 minutes. After sterilization the vessel iscooled and 50 μg/mL of kanamycin and 50 μg/mL chloramphenicol areaseptically added to the fermentation broth. The fermentation controlpoints are set at 37° C., pH 7.0, 35% dissolved oxygen, 8 SLPM offiltered dry air, and 100-600 rpm agitation. The agitation rate was usedto control the dissolved oxygen and the temperature was controlled byvarying the rate of cooling water to the fermenter. The vessel wasallowed to equilibrate in temperature, dissolved oxygen, pH and otherchemical characteristics. The fermentation vessel was then inoculatedwith 1% v/v of the starter culture. The OD 600 of the fermentation brothwas regularly monitored by taking a sample from the fermentation vesselwhile maintaining the sterility of the vessel. A Pharmacia Ultrospec IIIspectrophotometer was used to measure light transmission at 600 nm. Whenthe OD 600 surpasses a value of 3.5 the E. coli was induced to produceOPH through the sterile addition of 47.7 mg/L IPTG and 13.0 mg/L CoCl₂.The induced fermentation was allowed to proceed for 4 hours at whichpoint the broth was harvested. The harvested volume from the fermenterwas approximately 7.75 liters.

The harvested broth was concentrated 5-fold through microfiltration anddiafiltration with PBS through a 0.14 μm ceramic microfilter. Thediafiltration through the microfilter removed soluble salts and polymersfrom the solution. The enzyme is contained in the E. coli cells andhence is retained by the microfilter. The filter permeate was analyzedto determine that little enzyme is lost in this filtration. The finalvolume of concentrated microfiltered cells was approximately 1.5 liters.The concentrated broth was then centrifuged in aliquots of approximately50 mL at 3000×G for 30 minutes and the supernatant was removed. The cellpellets from this process were stored at −20° C. until needed.

Example 1 Cell Immobilization Procedure using PEI and Glutaraldehyde

For immobilization processes one or more of the frozen pellets wasremoved from the freezer and allowed to thaw. 10 g of the thawed cellpellet was resuspended in 100 mL of pH 7.0 phosphate buffer solution(PBS). Glutaraldehyde (GA) was added slowly to this volume over a periodof 1 minute to a final concentration of 0.2% (v/v) and mixed for onehour at room temperature. The solution was then diluted with two volumesof deionized water to a total volume of approximately 300 ml and varyingamounts up to 0.075% (v/v) polyethylenimine (PEI) was slowly drippedinto the solution to flocculate the cells. Immobilized cell mass wascollected by centrifugation at 750×G for 10 minutes yieldingapproximately 10 grams of wet immobilized cells. The resulting solidswere spread out and dried at room temperature for 24 hours. The driedmaterial was then collected and mechanically ground to the desiredparticle size using a mortar and pestle or wet milling by glass beads ina test tube. If necessary these particles were again dried.

Different variations of this process are possible including the order ofaddition of PEI and GA, the sequential addition of PEI and GAintermittently, the time between additions, the simultaneous addition ofPEI and GA, the amounts and concentrations of PEI and GA added, etc. Inthe first variation in the process, the order of addition of PEI and GAwas inverted, although the same total concentration of these materialswas maintained. Otherwise the immobilization was performed using thesame procedures. A second immobilization variation from the originalinvolved using twice as much GA which was added in the same time andreacted for the one hour as before. The third variation from theoriginal immobilization procedure used one-half of the original amountof PEI. In yet another variation, GA could be replaced with any suitabledi-aldehyde.

Example 2 Biodegradable Immobilized Enzymes

The immobilization discussed above involved whole cells, PEI, andglutaraldehyde. The whole cells and the glutaraldehyde are biodegradablebut the PEI is not. In some applications, it would be desirable to havethe entire immobilized particle to be biodegradable. Therefore, PETcould be replaced with another, more biodegradable material. Materialssuitable could include biodegradable polymers with chemical groups thatcan react with glutaraldehyde such as proteins, gelatins, wool, chitins,chitosan, and the like.

For the formation of a biodegradable whole cell immobilized enzyme, theimmobilization process above was carried out with chitosan (CHI)substituted for the PEI. For immobilization processes one or more of thefrozen pellets was removed from the freezer and allowed to thaw. 10 g ofthe thawed cell pellet was re-suspended in 100 mL of pH 7.0 PBS. GA wasadded slowly to this volume over a period of 1 minute to a finalconcentration of 0.2% (v/v) and mixed for one hour at room temperature.The solution was then diluted with two volumes of deionized water to atotal volume of approximately 300 ml and varying amounts up to 1 g ofCHI was slowly added to the solution to flocculate the cells.Immobilized cell mass was collected by centrifugation at 750×G for 10minutes yielding approximately 10 grams of wet immobilized cells. Theresulting solids were spread out and dried at room temperature for 24hours. The dried material was then collected and mechanically ground tothe desired particle size using a mortar and pestle or wet milling byglass beads in a test tube. If necessary these particles were againdried.

Example 3 Immobilization of Soluble OPH Enzyme

Sometimes an enzyme is available in a soluble form (e.g. the enzyme isexcreted by the microorganism producing the enzyme). A biodegradableimmobilized enzyme may be a desirable product using such a solubleenzyme. One advantage of whole cell immobilization is the inexpensivebiodegradable biomass from the organism which produced the enzyme. It isalso possible for example to form an immobilized enzyme by using thesoluble enzyme, biomass from another fermentation (often waste),chitosan, and glutaraldehyde to form an inexpensive biodegradableimmobilized enzyme.

Initial preparation of Soluble OPH Enzyme

Soluble OPH enzyme was harvested from the frozen cell pellets throughchemical lysis and chromatography process. Frozen cell pellets werethawed for 5 minutes in a room temperature water bath. Cells wereinitially lysed with 5 mL lysis buffer (98.8% YPER, 1% proteaseinhibitor, 0.2% DNAse I) per gram of cell pellet. The pellet wasloosened with a Teflon coated spatula and an additional 3-5 mL of lysisbuffer was used to rinse cell material off the spatula into the rest ofthe lysate. The lysate was then incubated on ice on an orbital shakerfor 40 minutes. Following the incubation the lysate was clarifiedthrough centrifugation for 20 minutes at 20,000×G and 4° C. Thesupernatant from the centrifugation was collected and stored at 4° C.until needed.

For the chromatography process, the 8 mL of lysate was added per 2 mL ofprepared NTA-Nickel resin in a centrifuge tube. The lysate/resin mixturewas incubated on an orbital shaker for 30 minutes at 4° C. Followingincubation the mixture was centrifuged at 800×G for 5 minutes and thesupernatant removed. 13 mL binding buffer (50 mM phosphate buffer, 500mM NaCl) per 2 mL resin was added to the column. The resin was gentlyresuspended and allowed to settle before centrifugation at 800×G for 5min. Again the supernatant was removed and 13 mL of wash buffer (30 mMimidazole, 50 mM phosphate buffer, 500 mM NaCl) per 2 mL resin wasadded. After resuspending the resin and allowing it to settle it wascentrifuged at 800×G for 5 min, and the supernatant was removed. Theprocess with the wash buffer was repeated one time. Following the washstep, 3 mL of elution buffer (250 mM imidazole, 50 mM phosphate buffer,500 mM NaCl) was added per 2 mL of resin. This final mixture wasincubated on an orbital shaker at room temperature for 10 minutes. Afterallowing the resin to settle it was centrifuged at 800×G for 5 min, thesupernatant (elution fraction) was collected and held at 4° C. untilneeded.

Soluble enzyme was concentrated in the elution fraction by centrifugalfiltration employing 10,000 kDa filters. This process also served toexchange the buffer solution containing the protein. 12 mL of elutionfraction was added to each filtration device and centrifuged at 5000×Gconcentrating the material to 1 mL 10 minutes). Permeate was removed andthe filtrate was diluted back to 12 mL with storage buffer (50 mMphosphate, 500 mM NaCl). This process was repeated two times upon whichthe filtrate was collected and stored at 4° C. until needed.

Immobilization Procedures Using Soluble OPH

For immobilization of the soluble enzyme, 0.5 mL of concentrated OPHfiltrate was diluted to 1.5 mL with Dl water. 6 mg of chitosan was addedto this solution and allowed to mix for 30 minutes. The solution wasthen further diluted to 5 mL with DI water and up to 12 of a 25% GAsolution was slowly dripped into this solution. After the reaction withGA, the material was allowed to gravimetrically settle for several hoursand ˜4 mL of the supernatant was carefully removed such that theimmobilized solids were left undisturbed. This material was stored at 4°C. until needed. A variant on this immobilization process involved theaddition of 165 mg of sterilized A. niger mycelium (−12% solids)following the addition of chitosan.

Example 4 Variations on Immobilization of OPH contained in Cells

Slurry containing OPH and related cells in 100 mL of PBS (pH 7.5) wasprepared. Glutaraldehyde was added and the slurry stirred for 1 hour atroom temperature. Deionized water (200 mL) was added, followed by theaddition of chitosan. Flocculation initiated and continued whilestirring was continued for several minutes. When flocculation wascomplete, the solid was isolated and dried using one of two processes.The first process isolated the material by transferring it to centrifugetubes, and centrifuging at 750-1000×G for about 10 minutes. Drying ofthe particulate mass was performed by air drying for 24 hours. Whendesired, pellets were formed from the moist solid before drying. Thealternate process took the flocculated product and transferred it tolyophilization tubes and lyophilized using standard techniques until adry product was obtained. Through either process powdered material wasobtained by mechanically grinding the dried material to the desiredsize.

Several immobilized OPH enzymes were prepared utilizing glutaraldehydeas the crosslinker, with supports including polyethylenimine, chitosan,polypyrrole, and a biomass which includes dry cells containing OPH. Eachprocedure began by suspending a quantity of OPH enzyme in a startingbuffer of phosphate buffered saline and the procedure was allowed tocontinue long enough for substantially complete flocculation of thefinal immobilized enzyme. Chart 1 below provides information related tothe preparation of these immobilized variations. Differences in theprocedures used in each example are noted in the chart below.

CHART 1 materials Cross- Bio- Reactive Starting Buffer Diluent BufferProcedure Enzyme linker Mass Polymer Vol. Vol. T ID (mg) (g) (g) (g) IDpH (mL) ID pH (mL) (° C.) Notes OPH-6 OPH - GA - EC - PEI - PBS 7.0 50DI 7.0 100 25 a, b, d, h 6.19 0.20 6.04 0.23 H₂O OPH-5 OPH - GA - EC -PEI - PBS 7.0 50 DI 7.0 100 25 a, c, d, i 5.97 0.20 3.24 0.23 H₂O OPH-8OPH - GA - EC - PEI - PBS 7.0 50 DI 7.0 100 25 a, b, d, h 5.83 0.20 3.160.11 H₂O OPH-4 OPH - GA - EC - CHI - PBS 7.0 50 DI 7.0 100 25 a, b, d, h11.13 0.40 6.04 0.23 H₂O OPH-7 OPH - GA - EC - CHI - PBS 7.0 50 DI 7.0100 25 a, b, d, j 4.14 0.20 2.25 0.23 H₂O OPH- OPH - GA - EC - CHI - PBS7.0 47 DI 7.0 94 25 a, b, d, j 10A 1.35 0.05 0.73 0.06 H₂O OPH- OPH -GA - EC - CHI - PBS 7.0 50 DI 7.0 100 25 a, b, d, j 11A 0.28 0.11 0.150.12 H₂O OPH- OPH - GA - EC - CHI - PBS 7.0 47 DI 7.0 94 25 a, b, d, j16A 1.30 0.05 0.71 0.11 H₂O OPH- OPH - GA - EC - CHI - PBS 7.0 50 DI 7.0100 25 a, b, d, j 16B 1.26 0.10 0.69 0.23 H₂O OPH- OPH - GA - EC - CHI -PBS 7.0 50 DI 7.0 100 25 a, b, d, j 15 4.00 0.20 2.17 0.69 H₂O OPH-OPH - GA - EC - CHI - PBS 7.0 47 DI 7.0 94 25 a, b, d, k 10B 1.24 0.100.68 0.10 H₂O PPY - 0.01 OPH- OPH - GA - EC - CHI - PBS 7.0 69 DI 7.0138 25 a, b, d, k 11B 1.52 0.15 0.83 0.08 H₂O PPY - 0.08 OPH- OPH - GA -EC - PPY - PBS 7.0 50 DI 7.0 100 25 a, b, d, l 14 2.52 0.20 1.37 0.23H₂O OPH- OPH - GA - EC - YE - 0.15 PBS 7.0 32 DI 7.0 64 25 a, b, d, m 3A0.84 0.07 0.46 H₂O OPH- OPH - GA - EC - PM - 0.15 PBS 7.0 32 DI 7.0 6425 a, b, d, n 3B 0.93 0.07 0.51 H₂O OPH- OPH - GA - EC - PBS 7.0 32 DI7.0 64 25 a, b, e, o 3C 0.76 0.07 0.41 H₂O AN - 0.11 OPH- OPH - GA -EC - CHI - PBS 7.0 32 DI 7.0 64 40 a, b, f, o 12A 0.77 0.07 0.42 0.08H₂O OPH- OPH - GA - EC - CHI - PBS 7.0 32 HEPES 8.0 64 5 a, g, o, i 9B0.58 0.12 0.32 0.08 OPH- OPH - GA - EC - CHI - PBS 4.0 32 DI 4.0 64 25a, g, o, i 12B 0.79 0.07 0.43 0.08 H₂O OPH- OPH - GA - EC - CHI - PBS10.0 32 DI 10.0 64 25 a, g, o, i 12C 0.72 0.07 0.39 0.15 H₂O OPH- OPH -DMP - EC - CHI - PBS 7.0 32 HEPES 8.0 64 25 a, g, o, p 9A 0.70 0.09 0.380.08 OPH- OPH - DSS - EC - CHI - PBS 7.0 32 HEPES 8.0 64 40 a, g, o, q9C 0.53 0.09 0.29 0.08 OPH- OPH - SA - EC - CHI - PBS 7.0 32 HEPES 8.064 5 a, g, o, r 1A 0.78 0.10 0.42 0.08 OPH- OPH - CC - EC - CHI - PBS7.0 32 HEPES 8.0 64 25 a, g, o, s 1B 212 0.17 1.15 0.08 OPH- OPH - DCH -EC - CHI - PBS 7.0 32 HEPES 8.0 64 25 a, g, o, t 1C 0.96 0.11 0.52 0.08Abbreviations OPH—organophosphorus hydrolase GA—glutaraldehyde EC—E.coli cells CHI—chitosan PPY—polypyrrole YE—yeast extract PM—pharmamediaAN—A. niger mycelium DMP—dimethyl pimelimidate DSS—disuccinimidylsuberate SA—succinic acid CC—cyanuric chloride DCH—1,6 diisocyantohexaneProcedural Notes a - suspend OPH and biosupport in starting buffer b -add GA and mix for about 1 hour c - add PEI and mix for about 1 hour d -add diluent to buffer solution e - add AN to diluent buffer and stir forabout 30 minutes f - add CHI to diluent buffer and stir for about 30minutes g - add CHI to diluent buffer h - slowly add PEI i - slowly addGA j - slowly add CHI k - slowly add CHI and PPY l - slowly add PPY m -slowly add YE n - slowly add PM o - slowly add diluent solution p -slowly add DMP q - slowly add DSS r - slowly add SA s - slowly add CCt - slowly add DCH

Additional Examples

A series of additional examples and tests were performed using a varietyof enzymes. The results of these examples and tests are summarized inthe charts and tables below. These examples are for illustrativepurposes only and are not intended to limit the scope of the disclosedtechnology in any way.

Example 5 Immobilization of Soluble Xylanase Enzyme

A slurry containing xylanase in 100 mL of PBS (pH 7.5) was prepared.Aspergillus niger and chitosan were added and the suspension stirred for1 hour at room temperature. Deionized water (200 mL) was added followedby the addition of glutaraldehyde. Flocculation initiated and continuedwhile stirring was continued for several minutes. When flocculation wascomplete, the solid was isolated and dried. Powdered material wasobtained by mechanically grinding the dried material to the desiredsize.

Unless otherwise noted, all of the examples listed in Chart 1 wereconducted at a temperature of 25° C. Each procedure began by suspendinga quantity of xylanase enzyme in a starting buffer of deionized waterand the procedure was allowed to continue long enough for substantiallycomplete flocculation of the final immobilized enzyme. Differences inthe procedures used in each example are noted in the chart below. Someimmobilization tests were performed without biomass in order to simplifyinvestigations of immobilization reaction conditions.

CHART 2 Materials Starting Diluent Bio- Reactive Buffer Buffer EnzymeCrosslinker Mass Polymer Vol. Vol. Procedure ID (mg) (g) (g) (g) pH (mL)ID pH (mL) Notes XY-1 XY - GA - 0.70 CHI - 7.0 50 DI 7.0 100 a, b, c 1001.20 XY-2 XY - GA - 0.70 AN - 10.00 7.0 50 DI 7.0 100 a, d, c 100 XY-3XY - GA - 0.70 CHI - 7.0 150 f, e 100 1.20 XY-4 XY - GA - 0.66 CHI - 4.0150 f, e 100 1.32 XY-5 XY - GA - 0.66 CHI - 10.0 150 f, e 100 1.32Abbreviations XY—xylanase GA—glutaraldehyde AN—A. niger myceliumCHI—chitosan DI—deionized water Procedural Notes a - add GA and let mixfor about 1 hour b - suspend CHI in diluent buffer c - slowly adddiluent solution to starting solution d - suspend AN in diluent buffere - slowly add GA to solution f - suspend CHI in starting buffer

Example 6 Immobilization of Soluble Peroxidase Enzyme

A slurry containing peroxidase in 100 mL of PBS (pH 7.5) was prepared.Chitosan was added and the suspension stirred for 15 minutes at roomtemperature. De-ionized water (50 mL) was added followed by the additionof glutaraldehyde. Flocculation initiated and continued while stirringwas continued for several minutes. When flocculation was complete, thesolid was isolated and dried using standard methods. Powdered materialwas obtained by mechanically grinding the dried material to the desiredsize.

Unless otherwise noted, all of the examples listed in Chart 4 wereconducted at a temperature of 25° C. Each procedure began by suspendinga, quantity of peroxidase enzyme derived from horseradish in a startingbuffer solution as noted in the chart, and the procedure was allowed tocontinue long enough for substantially complete flocculation of thefinal immobilized enzyme. Differences in the procedures used in eachexample are noted in the chart below. Several immobilizations wereperformed without biomass material in the composition to explore thedirect effectiveness of crosslinker materials on the enzyme.

CHART 3 Materials Reactive Starting Buffer Enzyme Crosslinker Bio-Polymer Vol. Procedure ID (mg) (g) Mass (g) (g) ID pH (mL) Notes PO-1PO - 9.9 GA - 0.66 CHI - 1.32 PBS 7.0 150 a, d PO-2 PO - 9.9 GA - 0.66AN - 0.66 CHI - 0.66 PBS 7.0 150 b, d PO-3 PO - 5.2 GA - 0.66 PPY - PBS7.0 150 c, d 0.26 PO-4 PO - 9.9 GA - 0.40 CHI - 1.88 PBS 7.0 150 a, dPO-5 PO - 10.1 DMP - 0.09 CHI - 1.81 HEPES 8.0 150 a, e PO-6 PO - 8.4DSS - 0.09 CHI - 1.81 HEPES 8.0 150 a, f PO-7 PO - 9.9 GA - 0.19 CHI -1.81 HEPES 8.0 150 a, d PO-8 PO - 8.0 SA - 0.1 CHI - 1.82 HEPES 8.0 150a, g PO-9 PO - 9.0 CC - 0.17 CHI - 1.83 HEPES 8.0 150 a, h PO-10 PO -9.7 DHC - 0.11 CHI - 1.80 HEPES 8.0 150 a, i Abbreviations PO—peroxidase(from horseradish) GA—glutaraldehyde DSS—disuccinimidyl suberateSA—succinic acid CC—cyanuric chloride PPY—polypyrrole DCH—1,6diisocyantohexane AN—A. niger mycelium CHI—chitosan DMP—dimethylpimelimidate Procedural Notes a - mix PO and CHI in starting buffer b -mix PO, AN, and CHI in starting buffer c - mix PO and PPY in startingbuffer d - slowly add GA to solution e - slowly add DMP to solution f -slowly add DSS to solution g - slowly add SA to solution h - slowly addCC to solution i - slowly add DCH to solution

Example 7 Immobilization of Soluble Alcohol Dehydrogenase Enzyme

A slurry containing alcohol dehydrogenase in 150 mL of PBS (pH 7.5) wasprepared. Chitosan was added and the suspension stirred for 15 minutesat room temperature. This was followed by the addition ofglutaraldehyde. Flocculation initiated and continued while stirring wascontinued for several minutes. When flocculation was complete, the solidwas isolated and dried.

Unless otherwise noted, all of the examples listed in Chart 2 wereconducted at a temperature of 25° C. Each procedure began by suspendinga quantity of alcohol dehydrogenase enzyme in a starting buffer ofphosphate buffered saline (pH=7.0) and the procedure was allowed tocontinue long enough for substantially complete flocculation of thefinal immobilized enzyme. In each example below where a diluent wasused, the diluent comprised 100 mL of deionized water having a pH of7.0. Differences in the procedures used in each example are noted in thechart below.

CHART 4 Materials Starting Reactive Buffer Enzyme Crosslinker Bio-Polymer Vol. ID (mg) (g) Mass (g) (g) (mL) Procedure AD-1 AD - 20.0 GA -0.66 CHI - 50 Suspend AD in Starting Buffer 1.32 Add GA and let mix for15 min Add Diluent Buffer to Starting solution Slowly add CHI Allow forcompletion of flocculation AD-2 AD - 20.0 GA - 0.66 AN - CHI - 50Suspend AD in Starting Buffer 0.66 0.66 Add ½ Diluent Buffer to Startingsolution. Slowly add GA, and stir for 30 min Add AN and CHI to remaininghalf of Diluent Buffer. Slowly mix Solutions Allow for completion offlocculation AD-3 AD - 17.5 GA - 0.66 EC - CHI - 50 Suspend AD and EC inStarting 0.46 1.01 Buffer Add CHI and let mix for about 15 min AddDiluent Buffer to Starting solution Slowly add GA Allow for completionof flocculation AD-4 AD - 19.9 GA - 0.66 EC - CHI - 50 Suspend AD and ECin Starting 0.38 1.00 Buffer Add CHI and let mix for about 15 min AddDiluent Buffer to Starting soln. Slowly add GA Allow for completion offlocculation AD-5 AD - 31.4 GA - 0.10 CHI - 150 Suspend AD and CHI inStarting 1.87 Buffer Stir till well mixed Slowly add GA Allow forcompletion of flocculation Abbreviations AD—alcohol dehydrogenaseGA—glutaraldehyde AN—A. niger mycelium CHI—chitosan EC—E. coli cells

Example 8 Immobilization of Soluble Pyruvate Decarboxylase Enzyme

A slurry containing alcohol pyruvate decarboxylase in 150 mL of HEPES(pH 7.5) was prepared. Chitosan was added and the suspension stirred forseveral minutes at room temperature. This was followed by the additionof glutaraldehyde. Flocculation initiated and continued while stirringwas continued for several minutes. When flocculation was complete, thesolid was isolated and dried.

Unless otherwise noted, all of the examples listed in Chart 3 wereconducted at a temperature of 25° C. Each procedure began by suspendinga quantity of pyruvate decarboxylase enzyme in a starting buffer ofphosphate buffered saline (pH=7.0) and the procedure was allowed tocontinue long enough for substantially complete flocculation of thefinal immobilized enzyme. Differences in the procedures used in eachexample are noted in the chart below.

CHART 5 Materials Starting Reactive Buffer Enzyme Crosslinker Bio-Polymer Vol. Procedure ID (mg) (g) Mass (g) (g) (mL) Preparation MethodPDC-1 PDC - 8.2 GA - 0.66 CHI - 1.32 150 Suspend PDC and CHI in StartingBuffer Stir till well mixed Slowly add GA Allow for completion offlocculation PDC-2 PDC - 8.1 GA - 0.66 AN - CHI - 0.66 150 Suspend PDC,AN, and CHI in 0.66 Starting Buffer Stir till well mixed Slowly add GAAllow for completion of flocculation PDC-3 PDC - 9.9 GA - 0.66 EC - 0.46CHI - 1.00 150 Suspend PDC, EC, and CHI in Starting Buffer Stir tillwell mixed Slowly add GA Allow for completion of flocculation PDC-4PDC - 10.7 GA - 0.66 EC - 0.38 CHI - 1.00 150 Suspend PDC, EC, and CHIin Starting Buffer Stir till well mixed Slowly add GA Allow forcompletion of flocculation PDC-5 PDC - 10.6 GA - 0.16 AN - 0.63 CHI -0.67 150 Wash AN in PBS Suspend PDC, AN, and CHI in Starting Buffer Stirtill well mixed Slowly add GA Allow for completion of flocculation PDC-6PDC - 10.9 GA - 0.16 AN - 0.65 CHI - 0.68 50 Suspend PDC and CHI inStarting Buffer Stir till well mixed Slowly add GA Mix for about 1 hourAdd 100 mL AN suspension Allow for completion of flocculationAbbreviations PDC—pyruvate decarboxylase GA—glutaraldehyde AN—A. nigermycelium CHI—chitosan EC—E. coli cells

Enzyme Activity Testing

One advantage of the immobilization is to increase the stability of theenzyme for temperature, solvent activity, and other improvements in thecharacteristics of the enzyme. To determine the improvements it isnecessary to measure the activity of the enzyme.

Organophosphate Hydrolase

OPH hydrolyzes many different organic compounds which containorthophosphate moieties such as the following list of compounds:acephate, coumaphos, demeton, diazinon, dursban, malathion, paraoxon,parathion, methyl paraoxon, and methyl parathion. The general reactionscheme is outlined as follows:

where X is either oxygen or sulfur, R and R′ are alkyl groups, and Z iseither an aryloxy group, a fluorine group, a thiol group, or a cyanide.It is understood that other methods may also be used to test enzymaticactivity, including examining an immobilized enzyme's ability tohydrolyze organic compounds other than those listed.

The activity of the enzyme defined as the number of phosphate bondsbroken per unit time depends greatly on the substrate. Paraoxon was usedas the substrate of choice for the enzyme activity measurement in thistest, although other substrates may also be used. The reaction has thefollowing stoichiometry.

The p-nitrophenol adsorbs light at 410 nm and hence the formation ofthis compound can be measured as a function of time. From thismeasurement it is possible to calculate the kinetics for the reaction.The rate is calculated from the linear section of the graph ofabsorbance due to p-nitrophenol vs. time. The selection of the linearsection can vary somewhat adding uncertainty to the estimate of thereaction rate.

Experimentally, an immobilized enzyme sample (10 milligrams) was addedto a substrate solution (12 mL) at a pH of 7.5 at room temperature andstirred.

Standard assay solutions consisted of 50 mM HEPES buffer, 0.1 mM CoCl₂,and 0.1 mM paraoxon in deionized water. The initial velocity of theenzyme is determined by sampling the assay solution and measuring theconcentration of the assay product versus time. Thus the concentrationof p-nitrophenol was determined through spectrophotometer measurementsat 410 nm every 3 minutes. The enzyme's activity was determined by thedecomposition of paraoxon (reported as (units/mg organophosphorushydrolase enzyme)). One unit results in the decomposition of 1.0 μmoleof paraoxon per minute at pH 7.5 at 25° C. Immobilized enzymes had aparticle size of about 200 p.m. Table 5 provides the OPH enzyme'sactivity. Other commonly accepted methods for determining enzymeactivity could also be used.

TABLE 1 Activity [units/g OPH] OPH-1A 17.9 OPH-1B 0.3 OPH-1C 11.1 OPH-3A7.1 OPH-3B 8.2 OPH-3C 7.4 OPH-4 1.8 OPH-5 1.5 OPH-6 0.7 OPH-7 2.7 OPH-81.2 OPH-9A 27.1 OPH-9B 59.9 OPH-9C 4.82 OPH-10A 5.1 OPH-10B 5.7 OPH-11A6.0 OPH-11B 2.9 OPH-12A 5.1 OPH-12B 7.1 OPH-12C 5.6 OPH-14 5.4 OPH-156.4 OPH-16A 5.8 OPH-16B 6.9

The solution was stirred during testing in order to reduce externaldiffusion influences. Diffusion will be a more significant limitation inthe immobilized enzyme compared to the whole cells from the fermenterbecause of immobilized cells particles are significantly larger than thewhole cells and hence the paraoxon has to diffuse further to reach theenzyme in the center of the immobilized particles. It may also bepossible to calculate the effect of the diffusion limitation but was notattempted in the present example. Reducing the particle size was alsoexamined as a method to reduce the influence of diffusion in theimmobilized particles.

FIGS. 1-2 show plots of absorbance vs. time for the whole cells from thefermentation and also for immobilized whole cells. It should be notedthat the presence of whole cells and the immobilized particles suspendedin solution interferes slightly with the absorbance even though most ofthe particles are settled before the absorbance was measured. The graphsshown in FIGS. 1-2 illustrate that a slower rate of reaction occurs withthe immobilized cells. Vigorous mixing has improved the rate of reactionfor the immobilized materials but better mixing conditions probably canbe found.

FIGS. 8-9 show two plots of absorbance vs. time for soluble OPH enzymeand also for variants of immobilized soluble enzyme. The assay for thesesolutions was the same as the one previously described for both freeenzyme and immobilized enzyme. The graphs show that the biodegradableimmobilized soluble enzyme can still accomplish the desired chemicalconversion. The data also demonstrates that the chitosan and A. nigerformulation has an improved activity compared to the chitosan onlyformulation as indicated by the higher reaction velocity.

Xylanase

Xylanases (XY) are enzymes that hydrolyse beta-1-4-xylane into xylose,thus breaking down the linear polysaccharide that makes up a significantportion of plant cell walls. The enzyme reaction has the followingstoichiometry:

Commercial applications for xylanase include uses in the papermakingprocess, as food additives in the processing of meats and breads, forthe clarification of fruit juices, and it is being explored for use inthe production of biofuel from plant material.

A colorimetric assay for evaluating the activity of xylanase involvesconverting xylan in a buffer solution at pH 4.5 at 37° C. for one hour.Following this reaction a sample is taken from the reaction solution andthe reducing sugars produced by the enzymatic reaction are furtherreacted with p-hydroxybenzoic acid hydrazide (PAHBAH) in a boilingalkaline solution which reacts with the reducing sugars to form aproduct that adsorbs at 410 nm. This absorbance can be compared to thatfrom a standard curve for reducing sugars and the activity of thexylanase can be calculated. Thus the enzyme's activity can be determinedby the amount of xylan converted to xylose (reported as (units/g)Xylanase enzyme). It was noted that the presence of Aspergillus nigermycelium appeared to elevate the xylose readings. Immobilized enzymeshad a particle size of about 200 μm. Table 2 provides the enzyme'sactivity ((units/g) immobilized Xylanase enzyme). Other commonlyaccepted methods for determining enzyme activity could also be used.

TABLE 2 Activity [units/g xylanase] XY-1 37.2 XY-2 31.6 XY-3 20.1 XY-412.6 XY-5 9.8

Alcohol Dehydrogenase

Alcohol dehydrogenases (ADH) are oxidoreductase enzymes that facilitatethe interconversion between a broad range of alcohols and aldehydes orketones with the reduction of nicotinamide adenine dinucleotide (β-NAD⁺to β-NADH). The enzyme reaction has the following stoichiometry:

where R represents alkyl groups although in some examples the alcoholinvolved is either a primary or secondary alcohol, or a hemi-acetals.ADH plays a role in the fermentation of alcohols by yeast and bacteriaand is key in the processing of ethanol in humans.

The activity of the enzyme is optionally defined by the conversion ofethanol to acetaldehyde. The assay for ADH was performed in whichstirred 12 mL assay solutions with 10-100 mg of immobilized enzyme orserial dilutions of 1 mg/mL enzyme stock solutions. The standard assaysolution consisted of a 50 mM sodium pyrophosphate buffer, 25% ethanol(v/v), and 4 mM β-NAD⁺ at a pH of 8.8. The velocity of the enzymereaction is determined by sampling the assay solution every 2 minutesand measuring the absorbance at 340 nm (corresponding to theconcentration of β-NADH).

The enzyme's activity was determined by the amount of β-NAD⁺ convertedto β-NADH (reported as units/g alcohol dehydrogenase enzyme). One unitconverts 1.0 μmole of ethanol to acetaldehyde per minute at pH 8.8 at25° C. Immobilized enzymes had a particle size of about 200 μm. Table 3provides the enzyme's activity. Other commonly accepted methods fordetermining enzyme activity could also be used.

TABLE 3 Activity [units/g alcohol dehydrogenase] AD-1 52.8 AD-2 93.3AD-3 27.6 AD-4 37.2 AD-5 27.3

Pyruvate Decarboxylase

Pyruvate decarboxylase (PDC) is a homotetrameric lyase (EC 4.1.1.1) thatcatalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbondioxide. The enzyme reaction follows the stoichiometry below:

where R is a representative alkyl group. PDC plays a significant role inthe anaerobic fermentation process in yeast.

The assay used to determine the activity of the PDC utilized an indirectmethod in which the conversion of pyruvate to acetaldehyde is linked tothe activity of ADH, which is provided in excess, and converts theacetaldehyde into β-NAD⁺ and ethanol. The reaction is monitored at 340nm, which corresponds to the consumption of β-NADH upon the formation ofacetaldehyde through the ADH driven reaction. For the assay animmobilized enzyme sample (˜10 milligrams) is added to a 12 mL solutionat pH 6.0 containing 200 mM citrate buffer, 34 mM sodium pyruvate, 0.1mM NAD⁺, and 35 U/mL of ADH. The velocity of the enzyme reaction isdetermined by sampling the sampling the assay solution and measuring theabsorbance at 340 nm every 3 minutes. The enzyme's activity wasdetermined by the amount of β-NADH converted to β-NAD (reported as(units/g pyruvate decarboxylase enzyme)). One unit converts 1.0 μmole ofpyruvate to acetaldehyde per minute at pH 6.0 at 25° C. Immobilizedenzymes had a particle size of about 200 μm. Table 4 provides theenzyme's activity. Other commonly accepted methods for determiningenzyme activity could also be used.

TABLE 4 Activity [units/g pyruvate decarboxylase] PDC-1 97.6 PDC-2 0.00PDC-3 78.7 PDC-4 87.8 PDC-5 18.6 PDC-6 20.2

Peroxidase

Peroxidases (PO) are a large family of enzymes that may catalyze thefollowing reaction:

ROOR′+2e ⁻(from an electron donor)+2H⁺(from a hydrogen donor)→ROH+R′OH

where R and R′ are from a wide variety of alkyl groups. For many ofthese enzymes the optimal electron donor is hydrogen peroxide, butothers have been shown to be more active with donors such as lipidperoxides. POs can be used industrially for the treatment of industrialwastewater and are being explored as an alternative to a number of harshchemicals that complicate manufacturing processes for adhesives,computer chips, and linings.

Peroxidase from horseradish was utilized in the case of these examples.The assay uses 4-aminoantipyrine as the hydrogen donor and hydrogenperoxide as the electron donor. The assay solution consists of 0.1 Mpotassium phosphate buffer, 0.85 mM hydrogen peroxide, 1.25 mM4-aminoantipyrine, and 85 mM phenol at a pH of 7.0.

An immobilized enzyme sample (10 milligrams) was added to the substratesolution (and stirred. The velocity of the reaction is monitored throughsampling the assay solution and monitoring the change in absorbanceevery 2 minutes at 510 nm as the reaction progresses. The enzyme'sactivity was determined by the decomposition of hydrogen peroxide(reported as (units/mg peroxidase enzyme)). One unit results in thedecomposition of 1.0 μmole of hydrogen peroxide per minute at pH 7.0 at25° C. Immobilized enzymes had a particle size of about 200 μm. Table 5provides the enzyme's activity. Other commonly accepted methods fordetermining enzyme activity could also be used.

TABLE 5 Activity [units/mg peroxidase] PO-1 9.7 PO-2 4.8 PO-3 19.3 PO-43.5 PO-5 6.1 PO-6 3.6 PO-7 1.9 PO-8 9.2 PO-9 2.9 PO-10 2.3

Enzyme Thermal Stability Testing

Many enzymes may lose activity over time and especially when thetemperature is elevated. Thermal stability is important for storageand/or usage when the enzyme can be exposed to higher temperatures.Enzymes were immobilized according to the process described above andused in the thermal stability tests. Determination of the long-termstorage stability under heated conditions of free and immobilized enzymewas made by exposing test tubes containing enzyme to 52.5° C., 65° C.,and 70° C. environments for periods from 30 minutes to 24 hours.Additionally effects on reactive pot-life can be determined by a similartest, the difference being that the enzyme material is mixed in asubstrate-free version of the assay solution during the heat exposure.For all evaluations, following exposure to heat the material was cooledin an ice bath for 10 minutes before the assay was run at roomtemperature.

Storage Temperature Testing

The graph shown in FIG. 3 shows the absorbance as a function of time fordifferent samples exposed to 52.5° C. for four hours. These samplesrepresent immobilized OPH samples at three different particle sizes andone free enzyme sample. It can be seen that the non-immobilized wholecell enzyme (“Free Enzyme” in the figure) has lost most of its activityafter four hours of exposure to 52.5° C. temperature whereas theimmobilized enzyme (“OPH” in the graph) retained significant activity.It can also be seen that the smaller sized particles give greateractivity as might be expected based upon mass transfer considerations.

Immobilized forms of OPH enzyme were tested for activity after beingsubjected to 65° C. for various periods of time. The results of thisexperiment are shown in FIGS. 7A and 7B. Similarly FIGS. 22A and 22Bshow the activity of PO in both immobilized and free enzyme forms afterexposure to 65° C. for similar time periods. The data demonstrates thatthis chitosan formulation has good temperature stability, similar to thePEI formulation.

The specific activity of enzyme material exposed to 65° C. for 0-120minutes was further explored as a means to simulate the storagestability of the immobilized enzyme and the change in activity (relativeto the activity of untreated material) with time at elevated temperatureis provided in Tables 6 A-C. Noteworthy is that the immobilizationprocess appears to increase the activity of the enzyme after exposure toelevated temperature. The presence of an increase in activity and itsdegree appear to be related to the formulation.

TABLE 6A Time at 65° C. 30 min 60 min 90 min 120 min Enzyme/Cell Slurry−79.1% −98.1% −99.2% −99.8% OPH-5 −27.4% −39.7% −22.4% −18.8% OPH-6+24.1% −9.6% +14.1% −16.6% OPH-7 −10.5% +0.9% −10.5% +20.3% OPH-8 −5.2%−14.9% −11.1% −2.3% OPH-9A — +0.4% — +8.3% OPH-9B — +17.0% — +34.7%OPH-12B — +2.1% — −1.4% OPH-16B — −8.3% — −1.0%

TABLE 6B Time at 65° C. 60 min 120 min Free XY Enzyme −0.7% +21.2% XY-1+31.8% +16.0% XY-2 +3.1% −35.6%

TABLE 6C Time at 65° C. 60 min 120 min Free PO Enzyme −7.5% −48.0% PO-1+22.8% −69.3% PO-3 −66.7% −63.2% PO-8 −10.5% −2.4%

Operational Temperature Testing

The relative decrease in specific activity, referenced to the unexposedcondition, of OPH (units/mg) of material exposed to 65° C. in 10% assaysolution for 0-120 min simulates operational half-life, and is providedin Tables 7 A-C. This data can be used to determine the extension of theenzyme's reactive half-life from immobilization, and its extent isdependent on the immobilization process and formulation.

TABLE 7A Time at 65° C. 60 min 120 min Enzyme/Cell Slurry −92.0% −98.2%OPH-6 −79.8% −80.3% OPH-7 −52.6% −77.9% OPH-9A −95.2% −93.0% OPH-9B−84.6% −85.3% OPH-12B −53.6% −73.6% OPH-14 −26.9% −45.7% OPH-15 −68.9%−80.9% OPH-16B −74.3% −86.6%

TABLE 7B Time at 65° C. 60 min 120 min Free Enzyme −54.2% −38.4% XY-1−23.2% −24.2% XY-2 +110.8% −38.8%

TABLE 7C Time at 65° C. 60 min 120 min Free PO Enzyme −49.1% −91.8% PO-1−85.4% −80.5% PO-3 −70.1% −83.1% PO-8 −84.2% −95.4%

Immobilized Enzyme Solvent Stability Testing

Many enzymes require the presence of water for activity in both the casewhere water is a component in the reaction (e.g. OPH catalyzedhydrolysis) and it can be involved in the enzyme configuration in itsactive state. However, enzyme activity is desirable on some chemicalsthat are sparingly soluble in water but more soluble in solvent-watermixtures. Therefore, it would be useful if enzyme activity could bemaintained in such solvents. Herein tests were performed measuring theimmobilized enzyme activity and immobilized activity in a variety ofsolvent-water solutions.

Isopropanol-Water Solution Stability

The enzyme's specific activity (units/mg) was initially studied inisopropanol/water solutions (10%, 20%) to determine the enzyme stabilityin water-soluble solvent solutions. The change in specific activity ofthe free and immobilized enzyme with exposure to isopropanol mixtures isprovided in Table 8, below. The percent change is referenced to theactivity of the sample in a 100% water solution.

TABLE 8 % isopropanol 10% 20% Enzyme/cell Slurry −69.6% −90.3% OPH-7−19.8% −56.5% OPH-14 −32.4% −70.0% OPH-15 −61.7% −84.4%

From this data it appears that the immobilization process does grantsome resistance to the denaturing action of the isopropanol compared tothe change in the activity of the free enzyme.

Methanol-Water Solution Stability

Following up on the initial isopropanol testing, the enzyme's specificactivity in methanol/water solutions (0%, 10%, and 20% methanol) wasstudied and the results are provided are reported as the change inactivity referenced to the activity of the material in a 100% watersolution in Table 9, below. This data shows that the performance of theimmobilized enzyme is related to the steps in the immobilizationprocess.

TABLE 9 10% Methanol 20% Methanol Free Enzyme/Cell Slurry −22.9% −78.9%OPH-4 −16.9% −37.2% OPH-5 −91.8% −88.7% OPH-6 −83.2% −54.8% OPH-8 −67.7%−92.6%

The absorbance vs. time for these experiments is shown in FIG. 4. As canbe seen in the graph, the activity for the free enzyme in 20% methanolis significantly reduced compared to the activity in 10% methanol. Thegraph shown in FIG. 5 shows the absorbance vs. time for two differentimmobilization, OPH-5 and OPH-6. These particles were screened with a30×30 mesh which provided particles of ˜200 μm equivalent diameter. Itcan be seen that the activity of the OPH-5 immobilized enzyme in the 10%and 20% methanol solution was fairly equivalent. In other words,increasing the methanol concentration in the solution did not reduce theenzyme activity of this immobilized enzyme.

The same experiments were performed with immobilization experimentsOPH-8 and OPH-6. These two immobilized enzymes were screened through twodifferent sizes meshes. The OPH-8 was ground to a particle size of about100 μm and the OPH-6 was ground to a size of roughly 60 μm diameter. Theresults of the experiment are shown in FIG. 6. It can be seen thatincreasing the methanol concentration from 10% to 20% reduces theactivity for the larger particles to a greater degree.

A variation on the experiments was performed as it was desired todetermine whether the activity of the enzyme would be reduced bycontinuous exposure to solvent over time. These experiments wereperformed with whole cell concentrates and immobilized particlesscreened with a 30×30 mesh from the OPH-7 immobilization. The activityassay performed is similar to the previous testing, except for thistesting the enzyme containing material was preexposed to 1.2 mL ofsolution consisting of 50 mM HEPES buffer in 10%, 20%, 30%, or 40%methanol. This pre-exposure lasted from 0-240 minutes and was followedby the addition of the 10.8 mL of an assay solution containing a similarconcentration of methanol and with the concentrations of HEPES, CoCl₂and paraoxon adjusted so that at the final volume of 12 mL the solutionwould match the concentrations of the standard assay solutionspreviously tested.

The enzyme's specific activity (units/mg) was studied in methanol/watersolutions (10%-40%) and the results are provided in Table 10, below.Time indicates how long enzyme material was exposed to solvent beforeassay was run. For each enzyme material the change is reverenced to thevalues at 10% methanol at 0 minutes of exposure.

TABLE 10 % Methanol 0 min 60 min 120 min 180 min 240 min Enzyme/ 10 0.0%8.1% 0.9% 7.1% 1.6% Cell 20 −57.8% −60.5% −69.1% −68.8% −67.0% Slurry 30−95.1% −93.4% −95.7% −94.8% −93.9% 40 −98.0% −98.1% −97.6% −97.6% −97.9%OPH-7 10 0.0% −3.2% 19.5% 15.7% 22.0% 20 −28.9% −44.6% −34.5% −42.3%−44.8% 30 −52.4% −65.4% −68.1% −70.3% −73.5% 40 −85.8% −95.9% −96.9%−97.3% −98.5%

From the data, the immobilized enzyme retains more activity over timewith exposure to methanol. Additionally the methanol seems to increasethe activity of the immobilized enzyme at the lowest testedconcentration. The charts shown in FIGS. 10-17 further illustrate, thatwhile the enzyme activity is reduced by the increased presence ofmethanol (MeOH), there are only slight changes with respect to theamount of time the enzyme is exposed to higher amounts of methanolsolution for both immobilized and whole cell OPH until the solutioncontains 30% or more MeOH. At this level a notable difference betweenimmobilized samples exposed to the solution for more than an hour ascompared to samples with no pre-exposure. Even with this decline inactivity, the immobilized material still appears to outperform the wholecells at similar MeOH concentrations as indicated by the degree ofchange in absorbance vs. time. As noted in other testing, the process ofimmobilizing the OPH containing cells gives the enzyme an increasedresistance to the deactivating effects of higher methanol concentrationsin the assay solution.

Ethylene Glycol-Water Solution Stability

The enzyme's specific activity (units/mg) was studied in ethyleneglycol-water solutions (10%-40% solvent), and the results are providedin Table 11, below. Time indicates how long enzyme material was exposedto solvent before assay was run. The listed change in activity in thetable is referenced to the activity for that particular enzyme form in a10% ethylene glycol solution at zero minutes.

TABLE 11 % Ethylene Glycol 0 min 60 min 120 min 180 min 240 min Free 100.0% 1.5% 0.9% 6.3% 5.2% Enzyme/ 20 −68.1% −62.5% −57.4% −69.0% −60.7%cell 30 −67.0% −55.3% −55.6% −59.9% −47.7% Slurry 40 −91.5% −87.3%−84.6% −80.9% −79.9% OPH-7 10 0.0% 29.0% 49.9% 38.2% 50.2% 20 −80.2%−53.6% −68.7% −71.3% −66.2% 30 −60.2% −72.8% −46.3% −51.3% −43.0% 40−71.3% −55.9% −70.3% −47.2% −46.5% OPH-14 10 0.0% 28.8% 77.5% 96.9%84.2% 20 −72.5% −73.7% −62.5% −65.4% −60.2% 30 −48.1% −53.7% −59.5%−53.2% −59.4% 40 −58.4% −68.5% −64.4% −72.4% −67.6% OPH-15 10 0.0% 0.0%49.8% 71.2% 100.5% 20 −74.0% −80.7% −70.4% −68.0% −66.1% 30 −64.4%−61.0% −60.4% −63.5% −62.3% 40 −85.2% −75.9% −76.4% −65.8% −68.0%

As with the isopropanol and methanol solution testing, the immobilizedforms of the enzyme appear to grant increased stability or resistance tothe denaturing properties of the solvent solution as compared to thefree enzyme. In some cases it appears that the immobilized forms undergoa transformation upon exposure to the solvent that increases the enzymeactivity as illustrated in FIGS. 18-21 which show the enzymatic activityrelative for each enzyme form relative to its activity in solvent-freesolutions.

Additional Solvents

The study of the enzyme's specific activity (units/mg) was expanded toinclude an additional aqueous solvent (DMSO) and to non-aqueous solvents(cyclohexane and toluene) in solution mixtures of 10%, 40%, 90%. Theperformance proportional to the enzyme form in solvent free solutions isprovided in Table 12, below.

TABLE 12 Solvent % Sample Solvent 10% 40% 90% Free Enzyme/Cell Slurrytoluene −72.3% −55.3% −87.4% OPH-7 toluene −34.8% −54.4% −1.1% OPH-16Btoluene −52.2% −72.9% −30.2% Free Enzyme/Cell Slurry cyclohexane −1.6%−69.8% −82.3% OPH-7 cyclohexane 90.2% 15.5% −16.0% OPH-16B cyclohexane28.1% −70.8% −85.4% Free Enzyme/Cell Slurry dimethyl −96.8% −97.9%−99.7% sulfoxide OPH-7 dimethyl 87.9% −56.9% −92.3% sulfoxide OPH-16Bdimethyl −6.0% −77.2% −97.2% sulfoxide

In general the immobilized forms perform better than the free enzyme insolutions containing these solvents. In a similar fashion to thepreviously tested solvents there appears to be some cases where theimmobilized enzyme activity increase with exposure to the solvent.

The Effect of pH

Related Studies at pH's Ranging from about 4.0 to 10.0

The enzyme's specific activity (units/mg) was studied (both as the freeenzyme and in the form of 200 μm particles) across a pH range of 4-10.The results, compared as the change in activity from standard assayconditions (pH 7.5), are provided in Table 13, below. Although theactivity is poor, the immobilized material does retain more activity inslightly acidic pH conditions (pH 5-6) compared to the free enzyme. Thechange in the activity of the immobilized enzyme is dependent on theformulation and process of immobilization, further exemplified bycomparing the change in specific activity (units/mg) with a shift of onepH unit in Table 14.

TABLE 13 pH Enzyme/Cell Slurry OPH-6 OPH-7 OPH-14 10 44.4% −34.2% 53.3%68.6% 9.5 58.1% −62.2% 147.2% 108.8% 9 82.8% −25.3% 78.3% 110.1% 8.576.1% −40.3% 90.8% 64.7% 8 84.0% −32.2% 89.1% 73.9% 7.5 0.0% 0.0% 0.0%0.0% 7 −33.0% −78.2% −18.3% −50.4% 6 −97.0% −77.9% −80.3% −81.8% 5−99.3% −55.9% −93.8% −96.5% 4 −99.6% −98.8% −97.9% −91.9%

TABLE 14 OPH- OPH- OPH- OPH- 10A 10B 11A 11B OPH-16A OPH-16B pH 7.5 5.15.7 6.0 2.9 5.8 6.9 pH 8.5 3.7 5.5 20.7 5.7 6.8 11.4 % Change −27.4%−4.6% 244.2% 100.9% 16.8% 64.7%

While the disclosed technology has been illustrated and described indetail in the drawings and foregoing description, the same is to beconsidered as illustrative and not restrictive in character. It isunderstood that the embodiments have been shown and described in theforegoing specification in satisfaction of the best mode and enablementrequirements. It is understood that one of ordinary skill in the artcould readily make a nigh-infinite number of insubstantial changes andmodifications to the above-described embodiments and that it would beimpractical to attempt to describe all such embodiment variations in thepresent specification. Accordingly, it is understood that all changesand modifications that come within the spirit of the disclosedtechnology are desired to be protected.

What is claimed is:
 1. An immobilized enzyme material comprising acrosslinked organophosphate-degrading enzyme having a support matrixthat includes a biomass material, wherein the crosslinkedorganophosphate-degrading enzyme was formed by reacting anorganophosphate-degrading enzyme with at least two polyfunctionalmaterials.
 2. The immobilized enzyme material of claim 1, wherein atleast one of the two polyfunctional materials is selected from the groupconsisting of a di-aldehyde, disuccinimidyl suberate, an organicdi-acid, glutaraldehyde, dimethyl pimelimidate, cyanuric chloride,succinic acid, hexamethylene diisocyanate, diimidoester, triazine,diisocyanate, and di(n-hydroxysuccinimide ester).
 3. The immobilizedenzyme material of claim 1, wherein at least one of the twopolyfunctional materials is a polyamine.
 4. The immobilized enzymematerial of claim 3, wherein at least one of the two polyfunctionalmaterials which is an polyamine is selected from the group consisting ofpolyethylenimine, polypyrrole, chitosan, a protein, and gelatin.
 5. Theimmobilized enzyme material of claim 4, wherein the biomass material isselected from the group consisting of bacterial cell material, fungalcell material, cellulose, dextran, starch agar, alginate, carrageenans,collagen, gelatin, albumin, ferritin, cotton, chitin, exoskeleton, andwool.
 6. The immobilized enzyme material of claim 1, wherein theimmobilized enzyme material is biodegradable.
 7. The immobilized enzymematerial of claim 1, wherein the biomass material is different from thebiomass material the organophosphate-degrading enzyme was derived from.8. The immobilized enzyme material of claim 1, wherein at least one ofthe two polyfunctional materials includes an additional biomass materialhaving a plurality of amine groups thereon.
 9. The immobilized enzymematerial of claim 8, wherein the at least one polyfunctional materialwhich includes a biomass material is selected from the group consistingof bacterial cell material, fungal cell material, cellulose, dextran,starch agar, alginate, carrageenans, collagen, gelatin, albumin,ferritin, cotton, chitin, exoskeleton, and wool.
 10. The immobilizedenzyme material of claim 1, wherein the biomass material is insoluble.11. The method of claim 1, wherein the organophosphate-degrading enzymeis selected from the group consisting of aryldialkylphosphatases (E.C.3.1.8.1) and diisopropyl-fluorophosphatase (E.C. 3.1.8.2).
 12. A methodfor preparing an immobilized organophosphate-degrading enzyme materialcomprising: providing a biomass material, an aqueous suspension of anorganophosphate-degrading enzyme, and at least two polyfunctionalmaterials; adding the biomass material, and one of the twopolyfunctional materials to the aqueous suspension of theorganophosphate-degrading enzyme to form a reaction mixture; adding thesecond polyfunctional material to the reaction mixture to flocculate theimmobilized organophosphate-degrading enzyme material.
 13. The method ofclaim 12, wherein the method further involves stirring the reactionmixture before adding the second polyfunctional material to the reactionmixture.
 14. The method of claim 12, wherein providing a biomassmaterial and the at least two polyfunctional materials involvesproviding a biodegradable biomass material and biodegradablepolyfunctional materials.
 15. The method of claim 12, wherein providinga biomass material involves providing a biomass material different fromthe biomass material that produced the enzyme.
 16. The method of claim12, wherein providing a biomass material involves providing a biomassmaterial is selected from the group consisting of bacterial cellmaterial, fungal cell material, cellulose, dextran, starch agar,alginate, carrageenans, collagen, gelatin, albumin, ferritin, cotton,chitin, exoskeleton, and wool.
 17. The method of claim 12, whereinproviding at least two polyfunctional materials involves providing anadditional biomass material having a plurality of amine groups thereon.18. The method of claim 12, wherein at least one of the twopolyfunctional materials is selected from the group consisting of adi-aldehyde, disuccinimidyl suberate, an organic di-acid,glutaraldehyde, dimethyl pimelimidate, cyanuric chloride, succinic acid,hexamethylene diisocyanate, diimidoester, triazine, diisocyanate, anddi(n-hydroxysuccinimide ester).
 19. The method of claim 12, whereinproviding at least two polyfunctional materials involves providing apolyamine selected from the group consisting of polyethylenimine,polypyrrole, chitosan, a protein, and gelatin.
 20. The method of claim19, wherein the biomass material is selected from the group consistingof bacterial cell material, fungal cell material, cellulose, dextran,starch agar, alginate, carrageenans, collagen, gelatin, albumin,ferritin, cotton, chitin, exoskeleton, and wool.
 21. The method of claim12, wherein the organophosphate-degrading enzyme is selected from thegroup consisting of aryldialkylphosphatases (E.C. 3.1.8.1) anddiisopropyl-fluorophosphatase (E.C. 3.1.8.2).
 22. The method of claim12, wherein providing a biomass material involves providing an insolublebiomass material.
 23. The method of claim 12, wherein at least one ofthe two polyfunctional materials is a polyamine.
 24. A method fordecontaminating an area containing a material susceptible to enzymaticdegradation comprising: providing a biodegradable immobilized enzymematerial in a form suitable for application to an area and adapted todegrade the material; contacting the area with the form of thebiodegradable immobilized enzyme material; wherein upon degradation ofthe material, the biodegradable immobilized enzyme material itselfbiodegrades, making its removal unnecessary.
 25. A method fortransforming a material susceptible to enzymatic transformationcomprising: providing a biodegradable immobilized enzyme materialadapted to transform the material in a form suitable for use in areaction chamber; placing the immobilized enzyme material in a reactionchamber; contacting the material to be transformed with the immobilizedenzyme material in the reaction chamber; removing the immobilized enzymematerial from the reaction chamber to an area where the immobilizedenzyme material may biodegrade.
 26. The method of claim 25, wherein thereaction chamber is a reaction column.
 27. The method of claim 25,wherein the reaction chamber is a closed reaction vessel.
 28. The methodof claim 25, wherein the reaction chamber is a packed bed.